The overall aim of the research described in this proposal is to establish the molecular mechanism of ATP hydrolysis catalyzed by F1- ATPase. The proposed studies will focus on the bovine heart mitochondrial F1-ATPase and the F1-ATPase from the thermophilic Bacillus PS3 (TF1) and functional complexes assembled from subunits of TF1. Inhibition and inactivation of MF1 and TF1 by amphipathic cations will be examined in detail. Covalent labeling of MF1 with quinacrine mustard and labeling of MF1 and TF1 with dequalinium suggest that amphipathic cations inhibit the enzymes by interacting with the C-terminal domains of alpha and beta subunits. Different sites are labeled when MF1 and TF1 are photoinactivated with dequalinium. Other amphipathic cations that inhibit MF1 at all concentrations tested, stimulate at ATPase activity of TF1 when present at low concentrations, but inhibit it at high concentrations. It is possible that the variation in minor subunits between MF1 and TF1 is responsible for the different responses of the two enzymes to amphipathic cations. To test this possibility, the MF1.OSCP complex will be photoinactivated with [14C]dequalinium and labeling sites will be determined. The sites labeled after photoinactivating the alpha3 beta3 gamma and alpha3 beta3 gamma delta complexes with [14C] dequalinium will also be determined. Syn C, a synthetic, amphipathic peptide, is a potent inhibitor of F1-ATPase. Three analogs of Syn C containing benzoylphenylalaine at different positions have been synthesized. All reversibly inhibit MF1 in the dark and also photoinactivate the enzyme. The sites labeled when TF1 and MF1 are photoinactivated with the Syn C analogs will be determined. A specific aim of the proposed research is to correlate kinetic parameters of F1-ATPases with the number of catalytic sites participating in ATP hydrolysis. To accomplish this, the kinetic characteristics of the ATPase activities of alpha3 beta3 gamma complexes assembled from TF1 subunits containing amino acid substitutions that affect activity will be examined in detail. Whether or not enzyme containing inhibitor MgADP trapped in a catalytic site is active or inactive is one problem to be resolved in these studies. Based on kinetic parameters, rules for vectorial catalysis coupled to ATP hydrolysis, and recent structural information, a reaction cycle is postulated for ATP hydrolysis under saturating conditions. Three catalytic sites are thought to be filled under these conditions. An integral feature of the proposed mechanism is ordered movement of the gamma subunit from a beta subunit to an adjacent alpha subunit after each hydrolytic step. Movement of the gamma subunit from the alpha subunit to a second beta subunit is postulated to poise the enzyme for another round of catalysis. A specific aim of the proposed research is to introduce cysteine residues at specific positions in the gamma subunit or alpha and beta subunits of TF1 for derivatizion with photoactivatable heterobifunctional cross-linkers. Irradiation of the modified complexes in the presence of inhibitors will produce different cross-linking depending on the inhibitor employed.